Full citations for references appear at the end of the examples section.
The use of proteins for use in a range of pharmaceutically related applications is subject to stringent regulatory requirements established by the Government. For example, in the U.S, the Center for Biologics Evaluation and Research (CBER) publishes a set of documents outlining the requirements regarding the production and monitoring of proteins produced transgenically (see fttp://www.fdagov/cber/cberftp/html), a similar set of regulations are related to the production and use of biologics in Canada. The CBER documents indicate that a biologic be produced from a reliable and continuous source, in order to ensure that a consistent product is obtained (ftp://ftp.fda.gov/cber/ptc/ptc.sub.-- mab.txt). This is because the product must be extensively tested and verified prior to its approval for use, and be available in the same form for future sales. There are several well established expression systems for the production of a biologic including master cell banks for cell culture, seed banks for transgenic plants, virus seed stocks for transgenic expression systems, and founder strains for transgenic animals. Master vector seed stocks must be generated for the use of transient expression systems, with the stability of the expression constructs routinely tested. If a protein such as a monoclonal antibody is to be produced from a cell line, documentation regarding the characterization of the parent cell line, cell production protocols, purification and quality control are required both of the master cell bank and working cell bank. Any changes to the manufacturing or formulation, especially if clinical trials are initiated require extensive re-characterization of the master and working cell banks and product, since these changes may result in significant changes of biological activity. It is stated within the CBER document "Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use" (Feb. 28, 1997), that "[i]t is recommended that the material used in the preclinical studies be manufactured using the same procedures as used or intended for use in manufacturing material for clinical trials". Furthermore, if any scale up is to take place, for example for Phase 2 studies, "that product comparability may have to be demonstrated . . . [which] may or may not require additional clinical studies" (ftp://ftp.fda.gov/cber/ptc/ptc.sub.-- mab.txt).
Plants have been used for the production of transgenic proteins, however, the same considerations that apply to the maintenance of master cell lines as discussed above, apply to the plant equivalent (see Miele, 1997). Seed banks for transgenic plants require periodic amplification since seeds can not be stored indefinitely. The storage of seed stocks must reduce potential genetic damage of the transgene of interest. Any other factors that may affect the seed bank must also be controlled including contamination of the seed by insects, fungi or bacteria. Furthermore, the stability of the transgene must be determined, as well as the levels of product expression in representative plants of a given seed lot, or between seed lots following reamplification of the seed bank. Additional data may also be required that compares the product prepared from different seed banks. This latter characterization also applies to year to year variations in harvested seed stocks. In general product development requires the constant monitoring of the biochemical and biological properties of the transgenic product (Miele, 1997). When these criteria are coupled to plant systems for the production of multimeric proteins the problem is compounded, since, in order to produce hybrid seeds capable of producing multimeric proteins, transgenic plants obtained from homologous seed lots, which require re-amplification and maintenance as outlined above, need to be crossed in order to produce the final hybrid seed, which again must be maintained as defined above. Clearly an alternate source of transgenic protein that is stable, and results in a continuous supply of protein without necessitating the extensive maintenance of multiples of seed banks is required.
The preparation of transgenic proteins with plants is well established within the literature and several successful transformation systems have been established. For example, multimeric proteins have been prepared via sexually crossing progeny of tobacco plants (Hiatt 1990, Hiatt and Pinney, 1992; Ma et al 1995), however, all plant sources to date that have been used for transgenic protein production for pharmaceutical applications have utilized annual plants. This necessitates the constant reamplification and reverification of seed bank stocks as described above. Clearly if an perennial plant species is utilized for the generation of transgenic proteins, the overall maintenance costs, and lot-to-lot variability would be greatly reduced. Furthermore, if vegetative structures of the perennial plant are harvested as a source of the transgenic protein, the regulatory requirements are reduced even more. In order to ensure the production of a perennial source of transgenic protein many factors must be taken into account that relate to the above requirements regarding reliability consistency of the product derived from a stable, continuous source.
One of the most important blood grouping reagents is the anti-human IgG reagent used for the detection of non-agglutinating antibodies. Mouse mAbs with suitable anti-human IgG specificity have been obtained and are gradually replacing the rabbit polyclonal anti-human IgG traditionally used in the Coombs' reagent (St Laurent et al 1993). These mAbs are produced by large-scale culture of B cell hybridomas. While reliable, this process is costly due to the need for sophisticated equipment, expensive culture media and trained personnel. In comparison with other diagnostic applications of mAbs, the market for proteins used in blood bank testing is highly competitive; consequently, prices for proteins are relatively low and the cost-efficiency of mAb production has become a critical issue.
The isolation of cDNA clones encoding the light and heavy chains of mAbs has allowed the expression of antibody genes in various heterologous systems including bacteria, fungi, insect cells, plants and non-lymphoid mammalian cells (Wang et al 1995; Wright et al 1992.). Amongst these systems, plants appear to be one of the most promising for cost-efficiency. However, following the initial demonstration in tobacco, it became important to find crop plants in which this technology could be brought to face modern demands for marketability. Hiatt et al (WO 96/21012, published Jul. 11, 1996) disclose methods for, and the preparation of therapeutic immunoglobulins ("protection proteins"), for use against mucosal pathogens, in plants. In EP 0 657 538, Galeffi and Natali disclose the production of antibodies for therapeutic or diagnostic use that recognize HER-2 oncogene present in mammary and ovary tumours. It is important to find crop plants in which recombinant proteins can be produced and that can face modem demands for marketability. These demands not only include competitivity of production costs, but also reliability, which implies that unless long-term stable supplies of the purified recombinant molecule can be established, means of insuring the perenniality of the homologated source material from which clonal populations can be derived must be developed. For B cell hybridomas, perenniality is insured by the establishment of a master cell bank, which consists of aliquots of cells taken from homogeneous pool and cryopreserved in liquid nitrogen. Hiatt (1990) suggests that alfalfa, soybean, tomato and potato may be useful alternatives as hosts for the propagation of antibodies. Alfalfa is one of the cheapest plant biomass to produce in current agro-ecosystems, and its perenniality in most climatic conditions makes it an attractive crop for sustainable agriculture. Furthermore, alfalfa (Medicago sativa L.) does not require annual tilling and planting, and the use of residual plant tissue for animal feed is well established (Austin and Bingham, 1997). However, several studies have examined the degree of proteolysis in ensiled forage species, and it known that proteolysis is more extensive in legume forages species than in grass species, with alfalfa exhibiting the highest rate and extent of proteolysis (Jones et al, 1995; Papadopoulos and McKersie, 1983). Furthermore, it is well known within the art that not all alfalfa plants are perennial, and of the perennial alfalfa plants not all are amenable to transformation protocols (Desgagnes, 1995).
The stability of transgenic proteins within plant tissues, and upon its extraction has been of concern in the literature. However, little is known about antibody stability in plant cell systems (Wongsamuth and Doran, 1997). Several workers (Hiatt et al (1989), During et al (1990), Ma et al (1995), Ma and Hein (1995), Schouten (1996)) have observed that chimeric constructs containing signal sequences for directing co-translational insertion of the constructs within the endoplasmic reticulum increase the stability of constructs within transgenic plants. In the absence of leader sequences, transgenic protein recovery is very low (Hiatt et al, 1989). It is a general practice to include protease inhibitors within the extraction cocktails in order to maximize protein recovery from transgenic plant tissues. However, marketable production of transgenic proteins from plants requires simplicity. For example, Austin and Bingham (1997) review large scale maceration and juice extraction protocols at the field site with final processing taking place at a processing plant several hours later. Such protocols employ the use of water and mechanical maceration and would be impractical if proteolysis within the extract was of concern, or if protease inhibitors were required during extraction. This is especially true for the application of alfalfa species known to exhibit high rates of proteolysis, especially after harvest (Jones et al 1995; Papadopoulos and McKersie 1983).
One kg of mAb has a present market value of $1,000,000 to $10,000,000. When production costs are estimated per gm of C5-1 produced under greenhouse conditions, including heating, manpower and consumables for extraction and purification, in a 250 m.sup.2 with an expected yield of 100 g per year, the cost per g of C5-1 would be between $500-$600, yet yield a market value of $400,000. Such estimates demonstrate that recombinant proteins could be produced cost-effectively in plants, however, a suitable plant system needs to be established. An aspect of an embodiment of this invention is directed to determining the characteristics required for a suitable transgenic plant line that can be used to produce a transgenic protein of interest that meets many of the criteria established within the CBER recommendations for a biologic compound.